The overall aim of this project is to determine by X-ray crystallography the precise molecular architecture of certain alpha-proteins that have dynamic as well as structural roles. Muscle proteins are a central focus and provide the background for studying related systems. A major aim is to characterize the strong actin-bound rigor conformation of myosin, as well to understand how myosin is regulated in scallop striated and (the closely related) vertebrate smooth muscles. All conventional myosin head (S1) crystal structures are thus far found in weak actin-binding states. Actin trimers and mini-thin filaments (MTF) are being prepared for crystallization in complex with S1 so that the strong actin binding state of the contractile cycle may be visualized. The trimers and MTFs will also be crystallized alone to provide a high resolution structure of undecorated F-actin. Structural investigations of myosin will also include studies of S1 with inhibitors of its ATPase activity, as well as various regions of the long myosin tail. Double headed constructs of myosin will be produced and studied in the """"""""off"""""""" state in order to understand myosin-linked regulation of contraction.
Our second aim i s to visualize atomic structures of the tropomyosin/troponin switch, which controls contraction in vertebrate striated muscles. These investigations will extend the recently completed structure determinations of the N- and C-termini of tropomyosin to the molecule's middle segments;also, novel peptide constructs will be used to obtain the structure of the critical head-tail overlap region that allows the filament to form.
The third aim, in a related area, is the analysis of fibrinogen/fibrin assembly. New central fragments of the molecule will be used in attempts to crystallize the DDE (-like) ternary nucleus of fibrin. Another goal is the structure determination of fibrinogen variants that underlie certain clotting disorders.
A final aim i s the analysis of design motifs in the alpha-helical coiled coil, especially in large fibrous proteins whose structures we are determining. We are convinced that detailed knowledge of the molecular mechanisms of muscle contraction and of the Ca2+-controlled tropomyosin/troponin switch is essential in order to correct malfunctions in various muscle diseases. Without atomic structures of these muscle proteins in different physiological states, the significance of disease producing mutations cannot be understood, nor can there be rational intervention at the molecular level directed to overcoming functional defects in these molecules. Similarly, in order to understand and control both normal and impaired clot formation, it is essential to have detailed structural information about the molecules and their interactions in the clot. A deeper understanding of the factors controlling the conformation, stability and partner selection in alpha-helical coiled coils will allow the design of therapeutic and diagnostic peptides targeted to naturally occurring coiled coil motifs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR017346-36
Application #
7590371
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Nuckolls, Glen H
Project Start
1974-09-01
Project End
2010-07-01
Budget Start
2009-03-01
Budget End
2010-07-01
Support Year
36
Fiscal Year
2009
Total Cost
$666,572
Indirect Cost
Name
Brandeis University
Department
Type
Organized Research Units
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Gillilan, Richard E; Kumar, V S Senthil; O'Neall-Hennessey, Elizabeth et al. (2013) X-ray solution scattering of squid heavy meromyosin: strengthening the evidence for an ancient compact off state. PLoS One 8:e81994
O'Neall-Hennessey, Elizabeth; Reshetnikova, Ludmila; Senthil Kumar, V S et al. (2013) Purification, crystallization and preliminary X-ray crystallographic analysis of squid heavy meromyosin. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:248-52
Brown, Jerry H (2013) Deriving how far structural information is transmitted through parallel homodimeric coiled-coils: a correlation analysis of helical staggers. Proteins 81:635-43
Kumar, V S Senthil; O'Neall-Hennessey, Elizabeth; Reshetnikova, Ludmila et al. (2011) Crystal structure of a phosphorylated light chain domain of scallop smooth-muscle myosin. Biophys J 101:2185-9
Brown, Jerry H; Kumar, V S Senthil; O'Neall-Hennessey, Elizabeth et al. (2011) Visualizing key hinges and a potential major source of compliance in the lever arm of myosin. Proc Natl Acad Sci U S A 108:114-9
Rosenbluth, Jack; Szent-Gyorgyi, Andrew G; Thompson, Joseph T (2010) The ultrastructure and contractile properties of a fast-acting, obliquely striated, myosin-regulated muscle: the funnel retractor of squids. J Exp Biol 213:2430-43
Brown, Jerry H (2010) How sequence directs bending in tropomyosin and other two-stranded alpha-helical coiled coils. Protein Sci 19:1366-75
Himmel, Daniel M; Mui, Suet; O'Neall-Hennessey, Elizabeth et al. (2009) The on-off switch in regulated myosins: different triggers but related mechanisms. J Mol Biol 394:496-505
Mentzer, Sarah E; Sundberg, John P; Awgulewitsch, Alexander et al. (2008) The mouse hairy ears mutation exhibits an extended growth (anagen) phase in hair follicles and altered Hoxc gene expression in the ears. Vet Dermatol 19:358-67
Brown, Jerry H; Yang, Yuting; Reshetnikova, Ludmilla et al. (2008) An unstable head-rod junction may promote folding into the compact off-state conformation of regulated myosins. J Mol Biol 375:1434-43

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